We have expanded the operational epitope map and functional map of the type 3 parainfluenza virus (PIV3) hemagglutinin-neuraminidase (HN) protein. Monoclonal antibodies (mAbs) to the HN protein define 14 operationally unique epitopes which are organized into 5 topographically non-overlapping antigenic sites (A, B, D, E, and F) and one bridging site (C). MAbs to sites A, B, and C inhibit hemagglutination and infectivity, and several site A mAbs also inhibit sialidase activity. MAbs to sites D, E, and F do not inhibit any known biological activity and react with all but 1 of 37 clinical PIV3 isolates examined, which is in contrast to mAbs to more variable epitopes in sites A, B, and C. Sequence analysis of HN genes of 16 mAb-resistant antigenic variants indicate the HN epitopes are located in hydrophilic stretches of amino acids. Computer analysis predicts these amino acids to form hydrophilic loops which connect B-sheet structures. Antigenic variants selected with mAbs which cross-react with the bovine PIV3 have amino acid substitutions in residues which are conserved in the primary structures of the parental human and bovine strains. MAbs to the fusion and G surface glycoproteins of respiratory syncytial virus have also been produced and are being characterized prior to selection of antigenic variants, epitope mapping, and sequence analysis of variants.